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Sheep brain glutathione reductase: Purification and general properties
Volume 250, number
1, 72-74
FEB 07243
June 1989
Sheep brain glutathione reductase: purification and general properties N.L. Acan and E.F. Texan Department
of Biochemistry,
Faculty of Medicine, Hacettepe
University, Ankara,
Turkey
Received 17 March 1989 Sheep brain glutathione reductase was purified about 1 1 000-fold with an overall yield of 40%. The method included ammonium sulphate fractionation, heat denaturation, 2’,5’-ADP Sepharose 4B and Sephadex G-200 chromatography steps. Specific activity at the final step was 193 IUjmg. The M, of the enzyme was found to be 116000 by gel filtration chromatography. On SDS-PAGE, two identical subunits of ,44, 64000 were obtained. From the spectral data, about 2 mol FAD per mol of enzyme were calculated. Glutathione reductase; Enzyme characterization; (Sheep brain)
1. INTRODUCTION
2. MATERIALS
GSSGR (NAD(P)H : oxidized glutathione oxidoreductase, EC 1.6.4.2) has been purified from a number of different organisms. The enzyme isolated from other sources has been reported to have an IV, value ranging between 100000 and 125000, consisting of two identical subunits, each containing an FAD ring [l-3]. The enzyme is responsible for maintaining glutathione in the reduced state. By coupling with the glutathione peroxidase reaction, GSSGR prevents cellular damage by oxidative stress. Brain is the organ with the highest risk of oxidative stress [4]. The aim of this study is to purify and characterize GSSGR from sheep brain, in which GSSGR is implied to have important functions.
2.1. Materials Fresh sheep brains were obtained from an Ankara slaughterhouse. All the other reagents were analytical grade.
Correspondence address: N.L. Acan, Department of Biochemistry, Faculty of Medicine, Hacettepe University, 06100 Ankara, Turkey Abbreviations: GSSGR, glutathione reductase; GSSG, oxidized glutathione; GSH, reduced glutathione
12
AND METHODS
2.2. Enzyme assay GSSGR assays were carried out spectrophotometrically, with a Beckman spectrophotometer model 25, at 37”C, modifying the method of Staal et al. [5]. The assay system contained 100 mM sodium phosphate buffer, pH 6.8, 5 mM EDTA, 1 mM GSSG and 0.12 mM NADPH. A unit of activity is defined as the oxidation of 1 pmol of NADPH per min under the assay conditions. 2.3. Protein assay Protein concentrations Bradford [6].
were determined by the method of
2.4. Purification of GSSGR Sheep brains were homogenized with 3 vols 50 mM sodium phosphate, pH 7.5, containing 1 mM EDTA (buffer A) and centrifuged at 14000 x g for 30 min. Ammonium sulphate fraction (35-55%) of the supernatant was dissolved in and dialyzed against the same buffer. The dialysate was heated for 1 h at 65°C. Precipitate was removed by centrifugation and the supernatant was applied to a column of 2’ ,5’-ADP Sepharose 4B (1 x 5 cm), equilibrated with buffer A. Activity was eluted with 10 ml of buffer A containing 1 mM NADPH and 1 mM GSH. Active fractions were loaded onto a column of Sephadex G-200 (1.6 x 75 cm) equilibrated with buffer A. The enzyme eluted
Published by Eisevier Science Publishers B. V. (Biomedical Division) 00145793/89/$3.50 0 1989 Federation of European Biochemical Societies
Volume 250, number 1
FEBS LETTERS
June 1989
Table 1 Summary of the purification steps Volume (ml)
Steps
14000 x g supernatant
Total protein (mg)
Specific activity (IWmg)
Purification (fold)
Yield (%)
500
4700
80.5
0.017
1
Ammonium sulphate fractionation (35-55%)
26
1872
58.5
0.031
1.82
Heat treatment (1 h at 65°C)
24.5
2 ’ ,5 ’ -ADP Sepharose 4B Sephadex G-200
46.3
SDS-polyacrylamide
57.1
6.3
0.38
35
31.6
0.17
31.9
was concentrated and stored at - 30°C after addition of 1 mg albumin per ml. 2.5.
Total activity (units)
gel electrophoresis
The method was carried out on 7.2% acrylamide slabs using modified Laemmli buffer system [7]. Coomassie Brilliant Blue R250 was used for staining. 2.6. Molecular mass determination Molecular mass of the enzyme was estimated by Sephadex G-200 gel filtration according to the method of Andrews [8].
3. RESULTS AND DISCUSSION The results of the purification are summarized in
1.233
100 73
72.5
72
92.592
5450
44
193.330
11355
40
table 1. In the 2’,5’-ADP Sepharose 4B step, NADPH was used for the elution of GSSGR. NADPH has been reported to be an inhibitor of the enzyme [2,9]. GSH was added to the buffer system to protect the enzyme. The aggregated forms of the erythrocyte enzyme have been reported especially upon exposure to NADPH, due to the oxidation of thiols at the active site [2]. For this reason, we thought that this effect was also overcome by the addition of GSH. Sephadex G-200 chromatography was used to eliminate NADPH completely (fig.1). The purified enzyme preserved as described retains its activity for about 2 months. The entire procedure of purification takes 4 working days. The molecular mass of the enzyme was found to be 116 kDa by gel filtration chromatography (fig.2). On SDS-PAGE, a single band was obtained. From the calibration curve, the subunit molecular mass was estimated to be 64 kDa (fig.3). The ratio of A280 to A460 was found to be 4.92.
\.Pyr’uvote
ktnose,
\qv,ne
dog muscle
albumin,
\
Lactate Bowne
trimer dehydrogenose. albumln. dimer
‘~\HexokinoSe,
0
Fig.1. Elution profile of Sephadex G-200 chromatography: (t?) enzyme activity; (G---O) AZ80 readings; (M), (M) A340 readings.
01
0.2
03
0.4
pg
muscle
yeast
0.5
K0V
Fig.2. Calibration curve of M, determination chromatography.
by gel filtration
73
Volume
250, number
FEBS LETTERS
1
65-
exl
4-
June 1989
Under the assay conditions, reaction is essentially in favour of reduction of GSSG. The reverse reaction was only 0.5% of that of the forward reaction. The enzyme also uses NADH with a specific activity of 8 IU/mg under the same conditions. Work on elucidating the kinetic properties and reaction mechanisms of the enzyme is now in progress.
3-
REFERENCES
II
0
I
0.2
0.6
0.4
0.8
1.0
R‘
Fig.3. Calibration
curve of subunit molecular mass determination by SDS-PAGE.
coefficient of the extinction Employing 11.3 mM_’ -cm-’ at 462 nm for enzyme-bound FAD [lo], 2.36 mol of FAD per mol of enzyme was calculated. These results are in agreement with those reported for GSSGR from other sources [l-3,5].
74
[l] Ii, I. and Sakai, H. (1974) Biochim. Biophys. Acta 350, 141-150. [2] Worthington, D.J. and Rosemeyer, M.A. (1974) Eur. J. Biochem. 48, 167-177. 131 Carlberg, I. and Mannervik, B. (1985) Methods Enzymol. 113, 484-490. [4] Orlawski, M. and Karkowsky, A. (1976) Int. Rev. Neurobiol. 19, 75-121. [5] Staal, G.E.J., Visser, J. and Weeger, C. (1969) Biochim. Biophys. Acta 185, 39-48. [6] Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. [7] Laemmli, U.K. (1970) Nature 227, 680-685. [S] Andrews, P. (1965) Biochem. J. 96, 595-605. [9] Mata, A.M., Pinto, M.C. and Lopez-Barea, J. (1985) Mol. Cell. Biochem. 67, 65-76. [lo] Massey, V. and Williams, C.H., jr (1965) J. Biol. Chem. 240, 4470-4480.
1, 72-74
FEB 07243
June 1989
Sheep brain glutathione reductase: purification and general properties N.L. Acan and E.F. Texan Department
of Biochemistry,
Faculty of Medicine, Hacettepe
University, Ankara,
Turkey
Received 17 March 1989 Sheep brain glutathione reductase was purified about 1 1 000-fold with an overall yield of 40%. The method included ammonium sulphate fractionation, heat denaturation, 2’,5’-ADP Sepharose 4B and Sephadex G-200 chromatography steps. Specific activity at the final step was 193 IUjmg. The M, of the enzyme was found to be 116000 by gel filtration chromatography. On SDS-PAGE, two identical subunits of ,44, 64000 were obtained. From the spectral data, about 2 mol FAD per mol of enzyme were calculated. Glutathione reductase; Enzyme characterization; (Sheep brain)
1. INTRODUCTION
2. MATERIALS
GSSGR (NAD(P)H : oxidized glutathione oxidoreductase, EC 1.6.4.2) has been purified from a number of different organisms. The enzyme isolated from other sources has been reported to have an IV, value ranging between 100000 and 125000, consisting of two identical subunits, each containing an FAD ring [l-3]. The enzyme is responsible for maintaining glutathione in the reduced state. By coupling with the glutathione peroxidase reaction, GSSGR prevents cellular damage by oxidative stress. Brain is the organ with the highest risk of oxidative stress [4]. The aim of this study is to purify and characterize GSSGR from sheep brain, in which GSSGR is implied to have important functions.
2.1. Materials Fresh sheep brains were obtained from an Ankara slaughterhouse. All the other reagents were analytical grade.
Correspondence address: N.L. Acan, Department of Biochemistry, Faculty of Medicine, Hacettepe University, 06100 Ankara, Turkey Abbreviations: GSSGR, glutathione reductase; GSSG, oxidized glutathione; GSH, reduced glutathione
12
AND METHODS
2.2. Enzyme assay GSSGR assays were carried out spectrophotometrically, with a Beckman spectrophotometer model 25, at 37”C, modifying the method of Staal et al. [5]. The assay system contained 100 mM sodium phosphate buffer, pH 6.8, 5 mM EDTA, 1 mM GSSG and 0.12 mM NADPH. A unit of activity is defined as the oxidation of 1 pmol of NADPH per min under the assay conditions. 2.3. Protein assay Protein concentrations Bradford [6].
were determined by the method of
2.4. Purification of GSSGR Sheep brains were homogenized with 3 vols 50 mM sodium phosphate, pH 7.5, containing 1 mM EDTA (buffer A) and centrifuged at 14000 x g for 30 min. Ammonium sulphate fraction (35-55%) of the supernatant was dissolved in and dialyzed against the same buffer. The dialysate was heated for 1 h at 65°C. Precipitate was removed by centrifugation and the supernatant was applied to a column of 2’ ,5’-ADP Sepharose 4B (1 x 5 cm), equilibrated with buffer A. Activity was eluted with 10 ml of buffer A containing 1 mM NADPH and 1 mM GSH. Active fractions were loaded onto a column of Sephadex G-200 (1.6 x 75 cm) equilibrated with buffer A. The enzyme eluted
Published by Eisevier Science Publishers B. V. (Biomedical Division) 00145793/89/$3.50 0 1989 Federation of European Biochemical Societies
Volume 250, number 1
FEBS LETTERS
June 1989
Table 1 Summary of the purification steps Volume (ml)
Steps
14000 x g supernatant
Total protein (mg)
Specific activity (IWmg)
Purification (fold)
Yield (%)
500
4700
80.5
0.017
1
Ammonium sulphate fractionation (35-55%)
26
1872
58.5
0.031
1.82
Heat treatment (1 h at 65°C)
24.5
2 ’ ,5 ’ -ADP Sepharose 4B Sephadex G-200
46.3
SDS-polyacrylamide
57.1
6.3
0.38
35
31.6
0.17
31.9
was concentrated and stored at - 30°C after addition of 1 mg albumin per ml. 2.5.
Total activity (units)
gel electrophoresis
The method was carried out on 7.2% acrylamide slabs using modified Laemmli buffer system [7]. Coomassie Brilliant Blue R250 was used for staining. 2.6. Molecular mass determination Molecular mass of the enzyme was estimated by Sephadex G-200 gel filtration according to the method of Andrews [8].
3. RESULTS AND DISCUSSION The results of the purification are summarized in
1.233
100 73
72.5
72
92.592
5450
44
193.330
11355
40
table 1. In the 2’,5’-ADP Sepharose 4B step, NADPH was used for the elution of GSSGR. NADPH has been reported to be an inhibitor of the enzyme [2,9]. GSH was added to the buffer system to protect the enzyme. The aggregated forms of the erythrocyte enzyme have been reported especially upon exposure to NADPH, due to the oxidation of thiols at the active site [2]. For this reason, we thought that this effect was also overcome by the addition of GSH. Sephadex G-200 chromatography was used to eliminate NADPH completely (fig.1). The purified enzyme preserved as described retains its activity for about 2 months. The entire procedure of purification takes 4 working days. The molecular mass of the enzyme was found to be 116 kDa by gel filtration chromatography (fig.2). On SDS-PAGE, a single band was obtained. From the calibration curve, the subunit molecular mass was estimated to be 64 kDa (fig.3). The ratio of A280 to A460 was found to be 4.92.
\.Pyr’uvote
ktnose,
\qv,ne
dog muscle
albumin,
\
Lactate Bowne
trimer dehydrogenose. albumln. dimer
‘~\HexokinoSe,
0
Fig.1. Elution profile of Sephadex G-200 chromatography: (t?) enzyme activity; (G---O) AZ80 readings; (M), (M) A340 readings.
01
0.2
03
0.4
pg
muscle
yeast
0.5
K0V
Fig.2. Calibration curve of M, determination chromatography.
by gel filtration
73
Volume
250, number
FEBS LETTERS
1
65-
exl
4-
June 1989
Under the assay conditions, reaction is essentially in favour of reduction of GSSG. The reverse reaction was only 0.5% of that of the forward reaction. The enzyme also uses NADH with a specific activity of 8 IU/mg under the same conditions. Work on elucidating the kinetic properties and reaction mechanisms of the enzyme is now in progress.
3-
REFERENCES
II
0
I
0.2
0.6
0.4
0.8
1.0
R‘
Fig.3. Calibration
curve of subunit molecular mass determination by SDS-PAGE.
coefficient of the extinction Employing 11.3 mM_’ -cm-’ at 462 nm for enzyme-bound FAD [lo], 2.36 mol of FAD per mol of enzyme was calculated. These results are in agreement with those reported for GSSGR from other sources [l-3,5].
74
[l] Ii, I. and Sakai, H. (1974) Biochim. Biophys. Acta 350, 141-150. [2] Worthington, D.J. and Rosemeyer, M.A. (1974) Eur. J. Biochem. 48, 167-177. 131 Carlberg, I. and Mannervik, B. (1985) Methods Enzymol. 113, 484-490. [4] Orlawski, M. and Karkowsky, A. (1976) Int. Rev. Neurobiol. 19, 75-121. [5] Staal, G.E.J., Visser, J. and Weeger, C. (1969) Biochim. Biophys. Acta 185, 39-48. [6] Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. [7] Laemmli, U.K. (1970) Nature 227, 680-685. [S] Andrews, P. (1965) Biochem. J. 96, 595-605. [9] Mata, A.M., Pinto, M.C. and Lopez-Barea, J. (1985) Mol. Cell. Biochem. 67, 65-76. [lo] Massey, V. and Williams, C.H., jr (1965) J. Biol. Chem. 240, 4470-4480.